Glutamic acid is the primary excitatory neurotransmitter in the brain. The broad representation of GLU in cortical and limbic pathways, its role in neurodegenerative processes, and the psychotomimetic effects of GLU receptor antagonists implicate GLU as an important neurotransmitter for understanding the pathobiology of psychiatric disorders. A dipeptide, N-acetyl aspartylglutamate (NAAG), appears to be an endogenous modulator of glutamatergic neurotransmission, occurs in high concentrations in brain, is colocalized to many forebrain glutamatergic neuronal systems and is released upon depolarization by Ca2+ dependent process. NAAG is an antagonist at NMDA receptors and activates mGLUR-3 receptors that inhibit GLU release. Furthermore, an enzyme designated N-acetylated alpha linked acidic dipeptidase (NAALADase), cleaves NAAG to GLU and N-acetyl aspartate (NAA) in brain. NAALADase appears to act as a fulcrum, determining the balance between NAAG inhibition of GLU and GLU liberated excitation. Reciprocal alterations in NAAG/NAALADase levels have been demonstrated in seizure models, neurodegenerative disorders and schizophrenia. During the previous period of support, cDNAs encoding for human prostate, human brain and rat brain NAALADase have been isolated and characterized. During the next period of support, we propose 1) to utilize site directed mutagenesis to identify which amino acids may be critical for the binding of Zn, the binding of substrate and the role of PKC phosphorylation sites. 2) Resolve isoforms of NAALADase to determine if different splice variants are differentially expressed in brain cell types. 3) Determine what factors may regulate NAALADase expression in the brain. 4) Use molecular strategies, including HSV transfection of sense and antisense and the construction of null mutations of NAALADase, to determine its role in glutamatergic neuronal function and behavior. 5) Purify and characterize the enzymes responsible for NAAG synthesis to begin to characterize presynaptic mechanisms regulating NAAG's availability. These studies should further clarify the role of NAAG in modulating brain glutamatergic neurotransmission that should have significant implications for neurodegenerative disorders, schizophrenia and epilepsy.